Endotoxin (ET) produced by the ubiquitous Gram-negative bacteria is an increasing concern in nephrology. Water for dialysis and dialysate are not sterile, and contain significant concentrations of bacteria and ET. During hemodialysis, the patient's blood is separated from the dialysate by a membrane whose molecular weight cut-off is about 20-40 kDa. In a dialysis session, 90-120 liters of dialysate are passed through the dialyzer. The most common dialysis regimen is 12 hours per week. The volume of dialysate used per patient per year is about 15,000 liters. According to existing water purity standards set by the AAMI, this translates into approximately 75 million endotoxin unit (EU) of ET and 30 billion bacteria passing through the "water side" of the dialyzer during one year of hemodialysis treatment. Recognizing that water systems are the main source of endotoxin contamination, the National Kidney Foundation recommends monthly LAL testing to monitor endotoxin levels in the water systems of dialysis centers.
Surveys of microbial and endotoxin contamination in reverse osmosis water and dialysate of different dialysis units of the U.S. and other countries frequently find colony forming unit (CFU) per ml and ET levels exceeding current AAMI standards. In a recent survey, 19-35% of water samples from dialysis centers had bacterial counts above the AAMI standards and 6% of dialysate samples exceeded the AAMI endotoxin limit (5 EU/ml water).
In the typical dialysis system, blood and dialysate are pumped into the dialyzer from opposite directions. At the inlet of dialysate, the hydrostatic pressure on water side can be higher than the hydrostatic pressure on the blood side. This facilitates the transfer of ET into the blood (backfiltration). ET also quickly adsorbs to the hydrophobic high-flux membranes. Displacement of adsorbed endotoxin by blood components may then take place.
In the first decades of artificial kidney treatment, technical efforts focused on developing effective dialysis membranes, machines and water systems. In the 1970s, some articles discussing the importance of pyrogenic reactions during hemodialysis induced installation of reverse osmosis system for preparation of more pure dialysis fluid. In the last decade, growing knowledge of the function of endothelial cells and their role in disease has helped to understand the possible alterations in endothelial cell structure and function evoked by uremia and its dialytic therapy.
Factors injuring the endothelium during hemodialysis include complement activation due to membrane contact, bacterial endotoxins, endotoxin containing immunocomplexes, hyperlipidemia, and cell adhesions. The activated monocytes migrate through endothelial intracellular junctions becoming macrophages; the filtered LDL particles transform them into foam cells. Bacterial endotoxin activates monocytes and the other white blood cells, increasing the chance for endothelial cell injury, arteriosclerosis and inflammatory problems such as amyloidosis.
Several articles discuss the significance of endotoxin transfer through dialysis membranes with cut-off points of 20-40 kDa. Komuro and Nakazawa have demonstrated that bacterial endotoxins in dialysates have two different molecular sizes, with one about 4 kDa (Komuro, T., Nakazawa, R.: Int. J. Artif. Organs (1993) 16:245-248). Urena and Herbelin (Urena, P., et al. Nephrol. Dial. Transplant. (1992) 7:16-28) used radiolabelled E. coli lipopolysaccharide (.sup.125 I M-LPS) to demonstrate that 7-10% of the radiolabelled lipopolysaccharide transferred from the dialysate to the blood side after a 4-hour recirculation period. The polysulfone (PS) and the polyacrylonitrile membranes (PAN) bound 10-17% of total initial activity of the radioactivity. The results suggest reduced LPS transfer across the Cuprophane (CU) membrane. The molecular weight of LPS units transferred through the membranes was &lt;43 kDa.
In similar experiments using Neisseria and Pseudomonas LPS, Laude-Sharp and Caroff demonstrated transmembrane passage of 1-8% of radiolabelled LPS (Laude-Sharp, M., et al.: Kidney Int. (1990) 38:1089-1094.). Between 5-70% of the radioactive LPS absorbed onto the membranes. PS exhibited the highest binding capacity for LPS. Vanholder and Haecke evaluated the dialysate and serum endotoxin concentrations in vivo before and after hemodialysis with small pore (PS400-F6, Fresenius, Germany) and large pore (PS600-F60, Fresenius, Germany) PS dialyzers. They detected significant Pseudomonas endotoxin transfer through PS600 membranes (Vanholder, R., et al.: Nephrol. Dial. Transplant. (1992) 7:333-339). The possible long-term consequences of cell stimulation and the subsequent release of inflammatory mediators such as Tumor Necrosis Factor alpha (TNF-alpha) and Interleukin-1 (IL-1) are a major concern. Use of sterile and endotoxin free dialysate significantly decreased the interleukin levels in patients' blood. Release of the endotoxin-binding monocyte receptor protein CD14 into the plasma was demonstrated in vivo during hemodialysis when the dialysate contained endotoxin (Bambauer, R., et al.: ASAIOTransactions (1990) 36, M317-320).
Dialysis amyloidosis is considered an inflammatory disease; the major protein of amyloid deposits is beta-2-microglobulin. Synthesis of this protein in macrophages is enhanced by endotoxins. Therefore, dialysis water contaminated with endotoxin may contribute to this process. Baz, et al. have shown that the onset of amyloidosis in long-term dialysis patients was considerably delayed when ultrapure dialysate was used (Baz, M., et al.: Int. J. Artif. Organs (1991) 14:681-685).
Many studies emphasize the importance of endotoxin-free dialysate and conclude that finding of transmembrane passage of low molecular weight intact species of LPS that are found in clinically used dialysates emphasizes the importance of obtaining LPS-free dialysates in order to improve the biocompatibility of hemodialysis (for a review, see Lonnemann, G. et al., Nephrol. Dial. Transplant. (1996) 11:946-949).
It is also becoming evident that besides endotoxin other pyrogenic substances must be removed when trying to prepare pyrogen-free water and dialysate for kidney dialysis. Klein et al. (Artif. Organs (1990) 14:85-94) have found that the primary bacterial species in contaminated water and dialysate was Pseudomonas. Pyrogenic substances originating from bacterial debris include LPS, peptidoglycans, muramyl peptides, exotoxin A and other substances yet to be identified. Of particular interest is a small (&lt;1000 dalton) cytokine-inducing fragment of exotoxin A found in bicarbonate dialysate contaminated with Pseudomonas aeruginosa (Mahiout, A. et al. J. Am. Soc. Nephrol. (1995) 6: 573). This low molecular weight, heat-stable, Pseudomonas pyrogen was partially purified and characterized (Massion, P. et al. J. Clin. Invest. (1994) 93: 26-32). There is a possibility that such a substance can penetrate even the tightest ultrafiltration membrane.
There are significant reasons to reduce pyrogen exposure of hemodialysis patients. The most acute is obviously to eliminate pyrogenic reactions. However, even more critical effects of long-term exposure to pyrogens include chronic leucocyte and monocyte activation, platelet activation, increased adhesiveness and aggregation, and complement activation which together with hyperlipidemia cause endothelium damage and lipid deposition in the arterial wall.
Efforts to produce more biocompatible dialysis membranes are hampered when pyrogenic substances are present during dialysis. The hemodialysis procedure by itself leads to activation of complement and coagulation proteins, platelets, leukocytes and the monocyte-macrophage system. When these effects are compounded by endotoxin, endothelium damage is increased. Hyperlipoproteinemia is also very common in patients with ESRD; together with the endothelium damage caused by endotoxin stimulus and extracorporeal treatment. It increases morbidity and mortality of the patient population from cardiovascular diseases. Endotoxin therefore, may significantly influence the long-term care and the cost of care in these patients due to cardiovascular complications.
This problem is very significant. In 1991, the gross mortality rate of chronic dialysis patients in the U.S. was over 20 percent, and in the active age population (&lt;55 years old) it exceeded 10 percent (Health Care Financing Research Report, End Stage Renal Disease (1991). Fifty percent of the patients died from cardiovascular diseases (Parfrey, P. S., Harnett, J. D.: ASAIOTransactions (1994) 40:121-129).
It has been demonstrated that bacterium and endotoxin-free dialysate resulted in reduced activation of monocytes and lower levels of interleukins and tumor necrosis factor in the patient's blood. Therefore, it is expected that regular use of sterile and endotoxin-free dialysate will help decrease the cardiovascular morbidity and mortality rate of patients undergoing hemodialysis. Since more than 50 percent of the patient population undergoing dialysis treatment is less than 65 years old, preserving their ability to work is very important. Procedures helping to slow the progression of cardiovascular diseases in patients undergoing hemodialysis will help decrease the cost of treatment and may improve the success of renal transplantation.
There is a need to develop a new technology for producing WFI quality water for dialysis without significantly affecting the cost of dialysis. The source water for dialysis is potable water. Even after treatment by the water companies, potable water-although safe to drink-contains low levels of mineral salts, trace metals, organic compounds, dissolved gases and colloidal matter, together with particles in suspension and microorganisms. Moreover, unlike other raw materials, water supplies vary widely in quality, both geographically and seasonally.
Before water can be used in the manufacture of biopharmaceuticals, it must be purged of its impurities to a degree which is defined by the pharmacopoeias and regulatory authorities like FDA. The most widely used and accepted method to produce WFI is distillation which makes WFI expensive. The quality of WFI is defined in terms of acceptable limits for inorganic and organic impurities determined by specific physical and chemical tests. WFI must be apyrogenic and free from suspended particles. The FDA recommends that the bacterial count should be below 10 CFU/100 ml. WFI must have a conductivity, measured on-line, less than 1.3 .mu.S/cm at 25.degree. C. However, the acceptable conductivity range of off-line samples-taking into account pH (which must lie between 5.0 and 7.0), temperature and carbon dioxide equilibrium-is likely to be 2.1 to 4.7 .mu.S/cm. The maximum acceptable total organic carbon (TOC) level is 500 parts per billion.
The modem approach to purifying water for bioprocessing applications is to use systems incorporating synergistic combinations of purification technologies. These technologies fall into two broad groups: ion-exchange and membrane processes. The major ion-exchange technique is deionization-using both cation-exchange and anion-exchange resins-while the principal membrane processes are reverse osmosis (RO), ultrafiltration (UF) and microfiltration (MF). The hybrid technology called electrodeionization (EDI) utilizes both ion-selective membranes and ion-exchange resins. These methods are then combined with distillation to produce WFI.
There are prior art methods describing the production of WFI without distillation. Reti and Benn (U.S. Pat. No. 4,610,790) disclosed a method using a plurality of filtration and deionization steps producing sterile water. Klein and Beach (U.S. Pat. No. 4,495,067) disclosed a similar system for making endotoxin-free water. We have to note, however, that despite these advances in membrane technology for endotoxin removal, distillation remained the preferred method for WFI. Another invention concerning endotoxin removal from biological fluids was disclosed by Harris (U.S. Pat. No. 3,959,128). He employed non-ionogenic hydrophobic synthetic polymers to adsorb endotoxin from biological fluids.
The literature quoted here points out the complexity of the spectrum of pyrogens present in water and dialysate. The discovery of the heat-stable, low molecular weight pyrogen(s) from Pseudomonas questions the efficacy of ultrafiltration as a tool for pyrogen removal. There is no evidence that the method of Harris would be useful in this regard either.
Hemodialysis requires high volumes of pure water at a low cost. The high cost of producing large volumes of WFI by the standard technique (distillation) precluded its use in hemodialysis even though it is warranted clinically. It would be desirable to develop a system capable of producing on-line WFI at a low cost from potable water in volumes necessary to meet the needs of hemodialysis units.